Peptide Nucleic Acids (PNAs) are synthetic analogs of DNA and RNA, first introduced in 1991 by Nielsen et al. at the University of Copenhagen. Unlike natural nucleic acids, PNAs feature a neutral polyamide backbone composed of N-(2-aminoethyl)glycine (AEG) units, to which nucleobases are attached via a methyl carbonyl linker. This structural modification replaces the sugar-phosphate backbone of DNA/RNA, conferring unique physicochemical properties such as high binding affinity, sequence specificity, and resistance to enzymatic degradation. PNA monomers are the fundamental building blocks for synthesizing PNA oligomers, and their design, synthesis, and modification are critical to the performance of PNA-based applications in molecular biology, diagnostics, and therapeutics.
1. Synthesis of PNA Monomers
PNA monomers are synthesized by combining an AEG backbone with a nucleobase acetic acid, followed by the incorporation of protecting groups to facilitate solid-phase peptide synthesis (SPPS). The synthesis process typically involves the following steps:
• Nucleobase Functionalization: The nucleobases (adenine, guanine, cytosine, thymine, or analogs) are alkylated at the N(2) position with bromoacetic acid to form nucleobase acetic acids. For adenine, cytosine, and guanine, the exocyclic amino groups are protected to prevent side reactions during synthesis.
• Backbone Preparation: The AEG backbone is synthesized, typically with a temporary protecting group on the primary amine, such as 9-fluorenylmethoxycarbonyl (Fmoc) or tert-butyloxycarbonyl (Boc).
• Coupling: The nucleobase acetic acid is coupled to the AEG backbone using coupling reagents like carbodiimides or benzotriazole uronium salts (e.g., HBTU) to form the PNA monomer.
• Purification and Characterization: Monomers are purified via high-performance liquid chromatography (HPLC) and characterized using techniques such as matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to ensure high purity (>97%).
2. Protecting Group Strategies
The choice of protecting groups is pivotal in PNA monomer synthesis, as it dictates compatibility with SPPS and influences solubility, coupling efficiency, and side reaction minimization. The three primary protection chemistries are:
• Fmoc/Bhoc Chemistry: The most widely used approach employs Fmoc for temporary protection of the AEG backbone’s primary amine and benzhydryloxycarbonyl (Bhoc) for permanent protection of nucleobase exocyclic amines. Fmoc is removed with 20% piperidine in dimethylformamide (DMF), while Bhoc is cleaved with trifluoroacetic acid (TFA) at the end of synthesis. This method is compatible with automated DNA synthesizers and allows the incorporation of sensitive reporter groups, but minor side reactions (0.3–0.4% contamination) can occur during Fmoc deprotection due to base-catalyzed rearrangements.
• Boc/Z Chemistry: The original method uses Boc for the backbone amine and benzyloxycarbonyl (Z) for nucleobases. Boc is removed with TFA, and Z is cleaved via hydrogenolysis or strong acid. This approach yields high-purity oligomers but is less compatible with DNA synthesizers due to the corrosive nature of TFA, limiting its use in mixed PNA-peptide syntheses.
• MMT/Base-Labile Chemistry: Monomethoxytrityl (MMT) and base-labile protecting groups are designed for PNA-DNA chimera synthesis, aligning with phosphoramidite chemistry. This method is less common but facilitates the preparation of chimeras with mixed nucleobase sequences.
Recent advancements include the use of N-benzoyl (Bz)-protected monomers, which are stable under Fmoc deprotection conditions and removable with aqueous ammonia, expanding the range of nucleobases (e.g., purines) for PNA-DNA chimeras. Additionally, Bts-based purine monomers without base-protecting groups have been developed, offering an atom-economical and environmentally friendly alternative by enhancing solubility without compromising coupling efficiency.
3. Modifications of PNA Monomers
To overcome limitations such as poor solubility, cellular uptake, and conformational flexibility, various modifications to PNA monomers have been explored:
• Chiral Monomers: Introducing chirality at the γ-position of the AEG backbone (γPNA) enhances binding affinity and sequence specificity. For instance, L-amino acid-derived γPNA monomers adopt a right-handed helical conformation, mimicking DNA, while D-amino acid-derived monomers form left-handed helices with reduced hybridization efficiency. Reductive amination and Mitsunobu coupling are common synthetic routes, with the latter yielding higher optical purity. Chiral monomers, such as those with lysine or alanine side chains, improve solubility and cellular uptake.
• Phosphonate-Conjugated Monomers: Phosphonate derivatives (e.g., glutamine phosphonate or lysine bis-phosphonate) introduce negative charges to the PNA backbone, enhancing solubility and enabling cationic lipid-mediated delivery. These monomers exhibit subnanomolar antisense activity in HeLa cells when delivered with lipofectamine, with activity increasing with the number of phosphonate units (e.g., P10-PNA with EC50 ~5 nM).
• Non-Natural Nucleobases: Modified nucleobases, such as 5-bromouracil, 5-methylcytosine, cyanuric acid, and G-clamp, enhance binding affinity or specificity. For example, 5-methylcytosine increases duplex stability in PNA-DNA chimeras, while cyanuric acid forms molecular tapes via hydrogen bonding, useful for structural studies.
• Lipophilic Conjugates: Conjugation with cholesterol or cholic acid improves cellular uptake but may reduce water solubility. These conjugates show nanomolar antisense activity (EC50 ~25 nM) when delivered with cationic lipids, maintaining high sequence specificity.
• Spacer Molecules: Hydrophilic spacers like AEEA (aminoethoxyethoxyacetic acid) are incorporated to improve solubility, especially in purine-rich sequences, and facilitate labeling with fluorophores or biotin.
4. Properties and Advantages
PNA monomers contribute to the following key properties of PNA oligomers:
• High Binding Affinity: The neutral backbone eliminates electrostatic repulsion, enabling stronger hybridization with DNA/RNA under low-salt conditions. PNA-DNA/RNA duplexes exhibit higher thermal stability than DNA-DNA/RNA duplexes.
• Sequence Specificity: Single-base mismatches significantly destabilize PNA-DNA/RNA duplexes, enhancing specificity. Two mismatches typically prevent hybridization.
• Enzymatic Stability: PNAs are resistant to nucleases and proteases, making them stable in vivo and in vitro, ideal for therapeutic and diagnostic applications.
• Chemical Stability: PNAs withstand a wide range of temperatures and pH conditions, ensuring robust performance in diverse environments.
• Versatility: PNA monomers support the synthesis of PNA-peptide conjugates and PNA-DNA chimeras, expanding their utility in molecular biology and bionanotechnology.
5. Challenges in PNA Monomer Synthesis and Use
Despite their advantages, PNA monomers face several challenges:
• Solubility Issues: Purine-rich sequences, particularly guanine-rich, tend to aggregate, reducing solubility. Spacer molecules and charged modifications (e.g., phosphonates) mitigate this issue but add synthetic complexity.
• Coupling Efficiency: Poor monomer solubility can lead to inefficient coupling during SPPS, limiting the synthesis of long PNA sequences. Double or triple coupling steps and higher monomer concentrations are often required.
• Side Reactions: Fmoc/Bhoc chemistry may result in minor base-catalyzed rearrangements during deprotection, though these are minimal (0.3–0.4% contamination). Boc/Z chemistry avoids some side reactions but is less compatible with modern synthesizers.
• Cellular Uptake: Unmodified PNAs exhibit poor cellular uptake due to their neutral backbone. Conjugation with lipophilic or charged groups and delivery systems like cationic lipids are necessary to enhance bioavailability.
• Optical Purity in Chiral Monomers: Epimerization during synthesis (e.g., via reductive amination) can reduce the conformational uniformity of γPNA, affecting hybridization efficiency. Mitsunobu coupling offers better stereospecificity.
6. Applications
The unique properties of PNA monomers have enabled a wide range of applications in molecular biology, diagnostics, and medicine.
• Antisense and Antigene Therapeutics: The ability of PNA to bind with high affinity to DNA and RNA allows it to be used as a steric blocker. In antisense applications, PNA targets and binds to a specific mRNA sequence, physically preventing the ribosome from translating it into a protein. In antigene applications, PNA can invade double-stranded DNA to inhibit transcription. This offers a powerful mechanism for controlling gene expression and has therapeutic potential for treating diseases driven by specific gene activity.
• Molecular Probes and Diagnostics: PNA probes are used for highly specific and sensitive detection of nucleic acid sequences. They are employed in methods like Fluorescence In Situ Hybridization (FISH) and are particularly useful for detecting single-nucleotide polymorphisms (SNPs) and gene mutations due to their excellent mismatch discrimination.
• Gene Editing and Genome Engineering: The strand invasion property of PNA is a crucial feature for non-enzymatic gene editing. PNA oligomers can be designed to target specific sites in the genome and facilitate homology-directed repair or other genetic modifications, providing an alternative to CRISPR-Cas9 systems.
• Antimicrobial and Antiviral Agents: PNA has been explored as a novel class of antimicrobial and antiviral agents. By targeting essential bacterial or viral genes, PNA can inhibit the synthesis of critical proteins, effectively stopping pathogen growth.
• Nanotechnology and Biosensors: PNA’s predictable binding properties and stability make it an ideal building block for self-assembling nanostructures. PNA-based systems are used to create biosensors that can detect specific nucleic acids with high sensitivity.
7. Recent Advances and Future Prospects
Recent developments have focused on improving PNA monomer synthesis and functionality:
• Atom-Economical Synthesis: Bts-based purine monomers without base-protecting groups reduce waste and enhance solubility, supporting large-scale PNA production.
• Chiral PNA Monomers: Advances in γPNA synthesis via Mitsunobu coupling improve optical purity, enhancing hybridization efficiency and enabling strand invasion of dsDNA/RNA.
• Novel Nucleobases: Incorporation of unnatural nucleobases like cyanuric acid and G-clamp expands PNA applications in structural biology and diagnostics.
• Tag-Assisted Liquid Phase Synthesis: This method substitutes solid supports with hydrophobic linkers, improving solubility and scalability for PNA synthesis.
Future research should address remaining challenges, such as optimizing coupling efficiency for long sequences, developing cost-effective synthesis methods, and improving cellular delivery without relying on cationic lipids. The exploration of novel backbone modifications, such as phosphono-PNA (pPNA) and chiral PNA analogs, holds promise for enhancing solubility and bioactivity. Additionally, integrating PNA monomers with nanotechnology and CRISPR-based systems could expand their therapeutic potential.
PNA monomers are the cornerstone of PNA technology, enabling the synthesis of versatile oligomers with applications in molecular biology, diagnostics, and therapeutics. Advances in protecting group strategies, chiral modifications, and novel nucleobases have significantly improved PNA performance, addressing issues like solubility and cellular uptake. However, challenges in coupling efficiency and scalability persist, necessitating further innovation. With ongoing research into atom-economical synthesis and advanced delivery systems, PNA monomers are poised to play a transformative role in biotechnology and medicine.
References:
1. PNA Monomers
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3. A Practical and Efficient Approach to PNA Monomers Compatible with Fmoc-Mediated Solid-Phase Synthesis Protocols
4. Synthesis of Peptide Nucleic Acid Monomers Containing the Four Natural Nucleobases: Thymine, Cytosine, Adenine, and Guanine and Their Oligomerization
5. Synthesis of (R)- and (S)-Fmoc-Protected Diethylene Glycol Gamma PNA Monomers with High Optical Purity
6. A convenient synthesis of chiral peptide nucleic acid (PNA) monomers
7. Synthesis and characterization of new chiral peptide nucleic acid (PNA) monomers
8. Modified PNA Monomers
9. Synthesis of New, Base-Modified PNA Monomers
10. PNA monomers fully compatible with standard Fmoc-based solid-phase synthesis of pseudocomplementary PNA
